Modern healthcare relies extensively on a range of chemical and biochemical analytical tests on a variety of body fluids to enable diagnosis, therapy and management of disease. Medical and technological advances have considerably expanded the scope of diagnostic testing over the past few decades. Moreover, an increasing understanding of the human body, together with the emergence of developing technologies, such as micro-systems and nanotechnology, are expected to have a profound impact on diagnostic technology.
Increasingly, diagnostic tests in hospitals are carried out at the point-of-care (PoC), in particular in situations where a rapid response is a prime consideration and therapeutic decisions have to be made quickly. Despite recent advances in PoC testing, several compelling needs remain unmet. For example, the detection of small molecules in biological samples is often very challenging, especially when no suitable receptor (e.g. enzyme, antibody, aptamer) with an appropriate specificity exists. The challenge is even greater when the molecule is lipophilic and a large proportion of the analyte is unavailable for analysis due to its association with hydrophobic components of the sample matrix such as cells, lipids and proteins.
The detection of lipophilic molecules in complex media (e.g. blood, plasma, saliva, urine, waste water and their extracts) is often difficult due to the association of the analyte with components of the sample matrix (e.g. plasma proteins and lipid membranes). The free, i.e. unbound, molecule concentration can be in the picomolar range and is often below the sensitivity limits of most commonly used measurement techniques, e.g. electrochemical, optical techniques. For this reason, state of the art methods for lipophilic molecule detection in complex media often involve intensive sample preparation, such as dilution/extraction of the sample into an organic solvent, centrifugation, evaporation and analysis by high pressure liquid chromatography (HPLC). Depending on the specific characteristics of the analyte molecule, post-HPLC column detection of the eluted compound is performed using electrochemical or optical methods such as absorption spectroscopy or fluorescence measurements.
The complex and time-consuming nature of HPLC assays for lipophilic molecules in complex samples mean that they are routinely performed by a very small number of specialist laboratories; for this reason the utility of these assays is rather limited. For example, for many lipophilic drugs, there is a clear need to develop alternative, miniaturised assays. This would enable real-time measurement and clinical intervention at the Point of Care (PoC).
Electrochemical techniques are often more amenable to Point of Care applications than optical ones, due to their lower cost and complexity. However, it is often difficult or impossible to detect lipophilic molecules in aqueous solutions using conventional electrochemical analysis. Reasons for these difficulties include, but are not limited to the fouling of the sensor (and concomitant loss of sensitivity) by surface adsorption of hydrophobic molecules and/or electrochemically generated reaction products; low analyte sensitivity; poor availability of the molecule in solution due to hydrophobic adsorption to components of the sample matrix such as proteins/lipids and a requirement for high oxidation/reduction potentials, thereby increasing the likelihood of other molecules in the sample interfering with the analyte signal.
Phenolic compounds, such as Propofol, are a good illustration of these challenges. Upon oxidation to phenoxy radical intermediates, the molecules can react with each other to form dimers, or can be further oxidised to quinones. Further oxidation cycles generate radical dimers and monomers, which react with each other to form a polymer. Repeated measurement often results in decreased sensitivity, caused by the electrically insulating polymer that builds up on the working electrode. In more complex biological samples, hydrophobic adsorption of components of the sample matrix (e.g. proteins, lipids and cells) to the working electrodes can also result in a severe loss of sensitivity. These problems mean that it is usually very difficult to develop a reliable assay for phenolic compounds, and other lipophilic compounds, in aqueous media.
Non-aqueous solvents are useful for electrochemical detection because the properties of these media can mitigate, or even eliminate, many of the problems described above. For example, the scarcity of protons in aprotic organic media, including Acetonitrile (MeCN), Dimethylsulfoxide (DMSO) and Dimethylformamide (DMF), means that free radical reaction products, such as the phenoxy radical, are much more stable, thereby simplifying the interpretation of the reaction system. Electrode fouling by hydrophobic adsorption is also eliminated through the use of these solvents.
However, not all the problems associated with measurement in water are avoided through the use of organic solvents. For example, some analytes still require high oxidation and reduction potentials for detection, and fouling can still occur, particularly for phenolic compounds such as Propofol. There is therefore a need to improve the reliability of detection and concentration measurement of lipophilic molecules in organic media, especially when the molecule is extracted into the organic media from a complex sample matrix, e.g. is derived from biological samples.
Many lipophilic molecules are potent free radical scavengers. It is possible to study the free radical scavenging ability of these molecules using electrochemical techniques. Most of the investigations presented to date rely on the electrochemical generation of the superoxide anion (O2.−). This free radical can be generated in aqueous or organic solvent by the electrochemical reduction of molecular oxygen or by solvation of potassium superoxide (KO2). The reactivity of O2.− differs in water and aprotic organic solvents. In aqueous environments, O2.−—acts as a strong nucleophile and spontaneously reacts with water to give hydro-peroxide and molecular oxygen:2O2.−+H2O→HO2−+O2+OH−
However, in aprotic media, O2.− is stable for up to 40 minutes and, depending on the species it reacts with, can act as a nucleophile, oxidant, reductant or base. The stability and diverse reactivity of this species in aprotic solvents has been exploited to measure the activity of lipophilic free radical scavengers, such as Probucol, Eblesen and tocopherols (Vitamin E). These assays measured the depletion of superoxide in the presence and absence of these free radical scavengers. A drawback of these assays is that they are not designed for use in clinical environments, e.g. for diagnostic purposes.